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Chapter 4

Nutrition and Shrimp Health
Wenbing Zhang and Kangsen Mai

Introduction

Animals consume foods to meet their physiological needs. Nutrients including protein (amino acids), lipids (fatty acids), carbohydrates (energy), vitamins and minerals, are essential for shrimp growth and health. It is generally accepted that nutrition is an important determinant of immune responses. The relation between infection and malnutrition is synergistic. Poor nutrition or poor feeding practices may lead to a reduced immune system response and lower the ability of shrimp to resist diseases. Therefore, quantitative and qualitative optimum nutrition play important roles in shrimp health. At present, nutritionists attach more importance in developing nutritional strategies that improve growth and production but do not necessarily optimize for disease resistance.

Immune system of shrimp is characterized by a lack of adaptive mechanisms mediated by antibodies. It is based on innate mechanisms consisting of cellular and humoral components that interplay to recognize and eliminate foreign microorganisms and pathogens. Cellular components include three types of hematocyte, i.e. hyaline, semi-granular and large granular cells (Aono et al., 1993). The humorals include prophenoloxidase (ProPO) system, superoxide dismutase (SOD), lysozyme (LSZ), antimicrobial peptides, reactvie oxygen species (ROS, e.g. O2-, H2O2 and OH-), hemocyanin, etc.

Recent years, research and development of immunostimulants, including some nutrients (e.g., vitamin C, n-3 highly unsaturated fatty acids), has increasingly attracted the attention of aquaculture scientists. An immunostimulant is a naturally occurring compound that modulates the immune system by increasing the host’s resistance against diseases that in most circumstances are caused by pathogens. According to Sakai (1999), immunostimulants can be divided into several groups depending on their sources: bacterial, algae-derived, animal-derived, nutritional factors as immunostimulants, and hormones/cytokines. The immunostimulatory effects of glucan, chitin, lactoferrin and levamisole for fish and shrimp have been reported. The most economical and feasible method of administration of immunostimulants to shrimp is by feeding as a supplement of feed. Therefore, we will also present a review of non-nutritional immunostimulants to shrimp in this chapter.

Protein and amino acids

Protein and amino acid requirements

The minimum dietary requirement for protein or a balanced mixture of amino acids is of primary concern in aquaculture because satisfying this requirement is necessary to ensure adequate growth and health of shrimp, while providing excessive levels is generally uneconomical, as protein is the most expensive dietary component. As such, most studies have been concerned with determining minimum dietary protein requirements of shrimp. Generally, recommended dietary protein levels vary from 30% to 55% for various species. The reported estimate of protein requirement must be carefully examined because it is dependent on quality (e.g., essential amino acid profile and digestibility) of dietary protein, age or physiological state of crustaceans, and environmental factors (e.g. salinity, dissolved oxygen, temperature).

Shrimp require the same 10 essential amino acids (EAA) as other species. The EAAs including arginine, methionine, valine, threonine, isoleucine, leucine, lysine, histidine, phenylalanine and tryptophan, were initially confirmed by Kanazawa and Teshima (1981). Generally, in contrast to the wide range of quantitative crude protein requirements that have been reported for shrimp, their qualitative requirements for amino acids are the same. This similarity provides a foundation for a theoretical approach to the nutritional causes of variation in optimal protein quality (D’Abramo et al., 1997).

Protein, amino acids and immune responses

A reduction in immune function could be expected if shrimp were fed a sub-optimal protein level in conjunction with nutritional stress. Pacific white shrimp Penaeus vannamei juveniles fed with a diet containing only 5% protein had reduced daily growth coefficient compared to shrimp fed diets with 40% protein (Pascual et al., 2004b). An increase in oxyhemocyanin was observed with increasing dietary protein levels indicating that shrimp accumulated protein as hemocyanin. A reduction of hemocytes occurred when shrimp were fed sub-optimal dietary protein levels indicating that zymogens contained in hemocytes, i.e., prophenoloxidase (ProPO) system, penaeidins and their activities (phagocytosis, coagulation), were also reduced. A reduction on respiratory burst was observed indicating that sub-optimal dietary protein level affected the number of cells and the phagocytosis capacity of cells (Pascual et al., 2004b).

Among the 10 EAAs, methionine, lysine and arginine are usually the most limiting amino acids for shrimp in commercial feed formulations and they are indispensable for normal growth and survival of shrimp (Coloso and Cruz, 1980; Pascual and Kanazawa, 1986; Akiyama et al., 1991). Shrimp P. vannamei fed methionine-, lysine- and arginine-deficient diets had significantly less wet weight gain than those fed the control diet, for the three amino acids, arginine appeared to be least limiting (Fox et al., 1995). However, arginine is involved in the synthesis of nitric oxide via nitric oxide synthase, which is inducible in shrimp (Jiang et al., 2004). Arginine has been shown to have numerous beneficial effects on T cell-mediated immunity in various animal models and in humans. Thus its involvement in shrimp health is certainly worthy of further investigation.

Lipids and fatty acid

Lipid and fatty acid requirement

Lipids are important components of shrimp diets because they provide a concentrated source of energy that is typically well utilized. In addition, dietary lipids supply the essential fatty acids (EFA) which cannot be synthesized by shrimp. Recommended lipid levels for commercial shrimp feeds range from 6% to 7.5% and a maximum level of 10% was suggested (Akiyama et al., 1991).

The unique aspect of lipid nutrition in penaeid shrimps is the requirement of polyunsaturated fatty acids, phospholipids and sterols. Adequate quantities of EFA must be provided in the diet to sustain normal growth and health of shrimp (NRC, 1993). A series of feeding experiments conducted by Kanazawa et al. (1977, 1978, 1979a,b) and Lim et al. (1997) demonstrated that four fatty acids are considered as essential for shrimp: linoleic (18:2n-6, LOA), linolenic (18:3n-3, LNA), eicosapentaenoic (20:5n-3, EPA), and decosahexaenoic (22:6n-3, DHA) acids, and the latter two n-3-highly unsaturated fatty acids (HUFA) are the most indispensable.

The deficiency of polyunsaturated fatty acids (PUFA) has been reported to reduce the survival rate, growth and feed conversion in P. vannamei (Lim et al., 1997), P. chinensis (Wang et al., 1993) and P. monodon (Merican and Shim, 1996). However, excessive dietary n-3 HUFA (?31.2 mg/g dry weight) may lead to detrimental effects on both the growth and survival of the postlarvae of P. monodon (Rees et al., 1994). The same thing occurs in P. vannamei (González-Félix et al., 2002).

Cholesterol and phospholipids

Cholesterol is a key sterol serving as a precursor to physiologically active compounds, including sex hormones, moulting hormones, adrenal corticoids, bile acids and vitamin D in shrimp (Sheen et al., 1994). It has been demonstrated that many shrimp are incapable of de novo synthesis of cholesterol from acetate. Therefore, dietary cholesterol is considered to be essential for good growth and survival. Based on the published data, requirements of dietary cholesterol in shrimp are 0.2-1.0%, e.g., juvenile shrimp P. japonicus (0.5%; Kanazawa et al., 1971), juvenile P. monodon (0.2-0.8%; Sheen et al., 1994), larvae P. monodon (1.0%; Paibulkichakul et al., 1998), P. vannamei (0.23-0.4%; Duerr and Walsh, 1996).

Phospholipids have been demonstrated to significantly affect survival, growth, deformities and/or resistance to stress in shrimp. They play a major role in maintaining the structure and function of cellular membranes, and act as emulsifiers in the gut and improve intestinal absorption of long chain fatty acids. Moreover, they stimulate lipoprotein synthesis in intestinal enterocytes and play an important role in the transport of dietary lipids.

There is a strong interaction between phospholipid and cholesterol. The optimal cholesterol requirement of P. vannamei decreased with the dietary phospholipid (de-oiled lecithin) increase. However, at the same dose of cholesterol, the more phospholipid in the diet, the faster growth could be found (Gong et al., 2000). This interaction was also observed in P. monodon (Paibulkichakul et al., 1998).

N-3 highly unsaturated fatty acid and immune responses

In mammals, it is known that dietary n-3 PUFAs are important precursors in the synthesis of eicosanoids, which in turn, are important mediators in the regulation of immune and inflammatory responses. Indeed, they markedly affect lymphocyte proliferation, natural killer cell activity, production of interleukin 2 (IL-2), and peripheral blood mononuclear cell proliferation. It is now generally accepted that n-3 HUFA, mainly eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are essential for growth, antioxidation, immunity, development and survival in marine fish.

Sheldon and Blazer (1991) found that macrophages from channel catfish fed an n-3 HUFA rich diet showed elevated bactericidal activity. Furthermore, rainbow trout Oncorhynchus mykiss fed n-3 PUFA-deficient diets were observed to be more susceptible to pathogens than fish fed diets supplemented with n-3 PUFA (Kiron et al., 1995). Wang et al. (2006) demonstrated that dietary n-3 HUFA increased immunity and resistance to Edwardsiella tarda challenge in Japanese flounder (Paralichthys olivaceus, Temminck and Schlegel). However, Erdal et al. (1991) reported decreased antibody titers and likelihood of survival in Atlantic salmon fed diets with high n-3 PUFA levels. Channel catfish fed a diet containing high n-3 fatty acid content had decreased survival following challenge with Edwardsiella ictaluri, compared to those consuming a diet with high n-6 fatty acid content (Li et al., 1994b). It is suggested that a carefully balanced dietary mixture of n-3 and n-6 fatty acids is needed to optimize the immune response.

There are still no direct data on effects of dietary n-3 HUFA on immunity and disease resistance ability in shrimp. Palacios et al. (2004) found that dietary n-3 HUFA improved the survival rate of P. vannamei postlarvae to low salinity. Moreover, they concluded that the beneficial effect of HUFA supplementation in the diet on survival to salinity stress test is partially related to modification of fatty acid composition of gills and to a larger gill area, which in turn enhances osmoregulatory mechanisms, namely Na+/K+-ATPase and carbonic anhydrase activities. Given the important links in vertebrate immune systems, further research with invertebrates is warranted.

Carbohydrates

Carbohydrate requirements and sources

The optimal dietary carbohydrate levels for shrimps are as follows: 26%, P. chinensis (Xu and Li, 1986); 21%, P. stylirostris (Rosas et al., 2000); 10-20%, P. vannamei (Guo et al., 2005).

In general, simple sugars are poorly utilized by shrimp. Sick and Andrews (1973) reported that growth and survival of P. duorarum fed diets containing 40% starch were higher than those fed diets containing 40% glucose. Alava and Pascual (1987) fed diets containing 10, 20 and 30% trehalose, sucrose or glucose to P. monodon, respectively. Shrimps fed diets containing trehalose and sucrose had higher weight gains and lower mortality than those fed the glucose diets. Shiau and Peng (1992) investigated the utilization of different carbohydrate sources and the possible protein-sparing effect by carbohydrate in P. monodon reared in seawater. In their study, three dietary protein levels (40, 35 and 30%) and three levels (20, 25, and 30%) of three carbohydrate sources (glucose, dextrin and starch) were tested. Results indicated that shrimp fed starch or dextrin had significantly higher weight gain, feed efficiency ratio, protein efficiency ratio and survival than those fed glucose. It also appears that starch has a better protein-sparing effect than dextrin or glucose. Accordingly, the required dietary protein level for P. monodon is lower if starch, instead of glucose or dextrin, is used as carbohydrate source. However, Pascual et al. (1983) fed diets containing maltose, sucrose, dextrin, molasses, cassava starch, corn starch or sago palm starch to P. monodon at levels of either 10 or 40% and found no correlation between survival and the relative complexity of carbohydrates.

Carbohydrate and immune responses

It is showed that blood protein and glucose of P. setiferus and P. vannamei juveniles are highly sensitive to dietary protein and carbohydrate (CHO) contents (Rosas et al., 2001a,b). Furthermore, P. vannamei and P. setiferus can convert protein to glycogen by the gluconeogenic pathway, which allows the shrimp to maintain a minimum circulating glucose independently from dietary CHO. Through protein metabolism, shrimp can synthesize their own CHO, regulate their osmotic pressure and glycogen synthesis, or store protein as hemocyanin. It has been demonstrated that blood glucose, triacylglycerols, cholesterol, and lactate together with blood protein, osmotic pressure, oxy hemocyanin (OxyHc), hemocytes and ProPO are good indicators of nutritional and immunological health in wild (Sánchez et al., 2001; Pascual et al., 2003) and cultivated shrimp (Rosas et al., 2001a).

Pascual et al. (2004a) investigated the effect of a size-based selection program on blood metabolites and immune response of P. vannamei juveniles fed different dietary carbohydrate levels. In this experiment, wild P. vannamei juveniles and a sample of seventh-generation cultured shrimp were acclimated under identical conditions. Shrimp were fed a high (HCHO: 44%) or a low (LCHO: 3%) carbohydrate diet for 55 days. Wild shrimp showed a direct relation between dietary CHO and lactate, protein and hemocyte levels indicating that dietary CHO was used for protein synthesis via transamination pathways. In seventh-generation cultured shrimp these parameters were inversely proportional to dietary CHO level, indicating the capacity to synthesize protein from dietary CHO was repressed in cultured shrimp. Farmed shrimp showed a limited capacity to respond to LCHO diets demonstrating high protein dependence in their metabolism and immune response. These results demonstrate that during size-based breeding programs other metabolic process than CHO catabolism can be selected. The incapacity of shrimp to use dietary CHO could limit protein reduction of diets and limit the efforts of the shrimp industry to be ecologically and environmentally profitable.

Vitamins

Dietary vitamin requirements

Vitamins are a heterogeneous group of organic compounds essential for the growth and maintenance of animal life. The majority of vitamins are not synthesized by the animal body or at a rate sufficient to meet the animal’s needs. They are distinct from the major food nutrients (proteins, lipids, and carbohydrates) in that they are not chemically related to one another, are present in very small quantities within animal and plant foodstuffs, and are required by the animal body in trace amounts. Although absolutely vital for various aspects of growth, health or reproduction, only minute amounts were required in the diet. This was explained by the idea that the role of these essential nutrients was limited to serving as co-factors for enzymatic reactions.

Despite an obvious physiological need by most shrimp for the vitamins, under practical farming conditions the quantitative dietary vitamin requirements will depend upon a number of important factors, including: feeding behaviour, vitamin synthesizing capacity of the gut microflora of the shrimp species, the intended culture system to be used (i.e. intensive, semi-intensive or extensive) and availability of natural food organisms within the water body, the size and growth rate of the shrimp species, the nutrient content of the diet, the manufacturing process, the physico-chemical characteristics of the water body and physiological condition of the shrimp species cultured.

Vitamin pathology

Vitamin deficiency

Vitamin deficiency is the result of many biochemical processes, beginning with low vitamin storage. Most of the vitamin related studies on shrimp concern the effects of supplementation on growth, feed efficiency, survival and biochemical indices. Information on vitamin deficiencies in shrimp is limited. The only well-documented vitamin deficiency in shrimp species is the black death syndrome related to vitamin C deficiency in penaeid shrimp. The following gross anatomical deficiency signs (Table 1) have been reported in shrimp fed vitamin deficient diets under controlled laboratory conditions.

Under intensive culture conditions, and in the absence of natural food organisms, dietary vitamin deficiencies may arise through: feed processing and storage; leaching of water soluble vitamins; deficiencies due to the presence of dietary anti-vitamin factors; deficiencies due to dietary antibiotic addition. The use of feed antibiotics to treat disease outbreaks may destroy the vitamin synthesizing capacity of the gut microflora of shrimp, which may play an important role in vitamin nutrition in some cases.

Vitamin toxicity

In contrast to the water soluble vitamins, shrimp accumulate fat soluble vitamins under conditions where dietary intake exceeds metabolic demand. Under certain circumstances

Table 1 Gross anatomical deficiency signs of vitamins in shrimp
Vitamin Deficiency signs Reference
Thiamin Anorexia, retarded growth, increased mortality rates, poor food conversion. Calculan et al., 1979; Deshimaru and Kuroki, 1979; Reddy et al., 1999
Riboflavin Light coloration, irritability, protuberant intersomite cuticle and short-head dwarfism. Chen and Wang, 1992
Pyridoxine Poor feed intake, growth depression, high mortality Giri et al., 1997; Reddy et al., 1999
Pantothenic acid High mortality, irritability, light coloration, soft and thin shell Shiau and Hsu, 1999; Reddy et al., 1999
Nicotinic acid

Anorexia, poor growth, poorer feed efficiency, blackening of the gills Reddy et al., 1999
Biotin

Anorexia, reduced percentage weight gain and food consumption Reddy et al., 1999
Folic acid

Decreased growth and food consumption Reddy et al., 1999
Cyanocobalamin

Retarded growth

Reddy et al., 1999
Inositol

High mortality

Kanazawa et al., 1976; Reddy et al., 1999; Shiau and Su, 2004
Choline Decreased growth, affected food consumption, anorexia, passive activity of the shrimp, low survival rate Reddy et al., 1999
Ascorbic acid

A general decline activity, poor food intake, anorexia, lesions in the abdominal region, black death syndrome Lightner et al., 1977; Magarelli et al., 1979; Li et al., 1994a; Li and Zhen 2005
Retinol Light coloration, pathological changes of visual tissue, deleterious effects on growth, malnourishment, degenerative changes in the hepatopancreas, poor survival Chen and Li, 1994; Reddy et al., 1999
Cholecalciferol Poor appetite, poor growth, softshell syndrome Li et al., 1994a; Chen and Li, 1995; Reddy et al., 1999
Tocopherol Poor growth, inappetence, darkening of the hepatopancreas, Reddy et al., 1999
Menadione No overt signs Shiau and Liu, 1994; Reddy et al., 1999

accumulation is such that a toxic condition (hypervitaminosis) may be produced. Although such a condition is unlikely to occur under practical farming conditions, hypervitaminosis has been experimentally induced. Toxicity signs are presented in Table 2.

Table 2. Toxicity signs of vitamins in shrimp
Vitamins Toxicity signs Reference
Cholecalciferol Inhibit absorb and accumulation of calcium and phosphorus Chen and Li, 1995
Riboflavin Reduced growth Xu et al., 1995
Nicotinic acid Reduced growth Xu et al., 1995
Pyridoxine Inhibition of protease activity Xu et al., 1995
Ascorbic acid

Low moult frequency, reduced growth

Guary et al., 1976

Vitamins and immune responses

Ascorbic acid

Ascorbic acid is thought to be important in optimal function of the immune system through enhancement of neutrophil production and also through protection against free radical damage. Ascorbic acid is present in high concentration in leukocytes and is utilized at a higher rate during infection and phagocytosis. Viral infections are known to cause reduction in serum ascorbic acid concentration. Furthermore, genetic selection and intensive aquaculture led animals to require ascorbic acid in their diet. Supplementation of feed with ascorbic acid gives help to restore normal levels, and thus, provide non-specific resistance against invading pathogens.

There has been considerable interest concerning the effect of dietary ascorbic acid on immune response and disease resistance in aquaculture animals especially in fish. A number of studies reported the improved immune responses and disease resistance in many fish species by feeding a higher level of dietary ascorbic acid than required for growth. Increased immunity was demonstrated by the increased immunological parameters, such as lysozyme, complement activities, phagocytic activity, and respiratory burst. For example, Ai et al. (2006) found that the activities of serum lysozyme and alternative complement pathway, phagocytosis percentage and respiratory burst activity of head kidney of large yellow croaker Pseudosciaena crocea increased with increasing dietary ascorbic acid. The challenge experiment with Vibrio harveyi showed that fish fed the diets with supplementation of ascorbic acid had lower cumulative mortality, and the cumulative mortality (16.7%) in fish with 489.0 mg/kg ascorbic acid was significantly lower than that (41.7%) in fish with 23.8 mg/kg ascorbic acid. This result coincide with those in channel catfish (Liu et al., 1989), Atlantic salmon (Hardie et al., 1991), turbot Scophthalmus maximus (Roberts et al., 1995), Japanese seabass Lateolabrax japonicus (Ai et al., 2004), grouper Epinephelus malabaricus (Lin and Shiau, 2005) and siberian sturgeon Acipenser baerii (Xie et al., 2006).

However, information about effects of dietary ascorbic acid on immune responses and disease resistance in shrimp is limited. Data from the research of Lee and Shiau (2002b) suggest that dietary ascorbate enhances immune responses in P. monodon and different ascorbate sources (L-ascorbyl-2-sulfate, L-ascorbyl-2- polyphosphate, L-ascorbyl-2-monophosphate-Na and L-ascorbyl-2-monophosphate- Mg) may affect the immune responses differently. Furthermore, Lee and Shiau (2003) found that increase of dietary ascorbic acid improved hemocyte respiratory burst response and growth, and prevented tissue Cu accumulation in P. monodon fed with high dietary Cu.

Tocopherol

Tocopherol functions as a lipid-soluble antioxidant, protecting biological membranes, lipoproteins and lipid stores against oxidation. The antioxidative functions of tocopherol include scavenging of free radicals to terminate lipid peroxidation, which can initiate damage to unstable intracellular components including membranes, nucleic acids and enzymes, and thereby result in pathological conditions. It is well established that a high dietary tocopherol supplementation enhances both humoral and cellular defences in mammals and fish. However, this information for crustaceans is limited. In the research of tocopherol requirements of juvenile grass shrimp P. monodon and effects on non-specific immune responses, Lee and Shiau (2004) concluded that the non-specific immune responses in shrimp improved as the level of tocopherol increased in the diet, reaching a peak after the requirement was met. The adequate dietary tocopherol requirement for maximal growth and maintenance of non-specific immune responses for growing P. monodon is about 85-89 mg/kg.

Minerals

General function and requirements

The main functions of minerals in the body include the formation of skeletal structure, maintenance of colloidal systems (osmotic pressure, viscosity, diffusion), and regulation of acid-base equilibrium. They are important components of hormones, enzymes, and activators of enzymes. A fixed number of specific trace metals (Fe, Mn, Cu, Co, Zn, Mo Se, etc) are firmly associated with a specific protein in metalloenzymes, which produce a unique catalytic function. Minerals are essential for the transmission of nerve impulses and muscle contraction.

With the exception of osmoregulation, the maintenance of osmotic balance between body fluids and the water in which the animal lives, the biochemical functions of minerals in aquatic species appear to be similar to those in terrestrial animals. Freshwater species lose ions to the hypotonic environment and therefore suffer from hydration, whereas the reverse is true for marine species. Unlike terrestrial animals, which are primarily limited to a dietary source of minerals, aquatic animals may be able to utilize, to some extent, minerals dissolved in the water to meet physiological requirements. Calcium, copper, iron, magnesium, sodium, potassium, selenium, and zinc are generally derived from the water to satisfy part of the requirements of shrimp. However, phosphates and sulfates are more effectively obtained from feed sources.

Mineral pathology

Mineral deficiency

Despite the adequate presence of macro and trace elements in virtually all raw ingredients commonly used for shrimp feeding, and the ability of shrimp to absorb certain trace elements from the surrounding water, mineral deficiencies may arise under intensive culture conditions.

Dietary imbalances may also reduce mineral bioavailability. The availability and utilization of dietary trace elements in shrimp is dependent upon the dietary source and form of the element ingested, the adequacy of stores within the body, interactions with other mineral elements present in the gastro-intestinal tract and within the body tissues (antagonisms), and finally by element interactions with other dietary ingredients or their metabolites (e.g., vitamins, fibre and phytic acid).

Mineral toxicity

A major hazard associated with the use of dietary feed ingredients is the presence of potentially toxic mineral elements such as the accumulative elements copper, lead, cadmium, mercury, arsenic, fluorine, selenium, molybdenum and vanadium. Feed ingredients that may contain potentially toxic metal contaminants include: fish meal (mercury, selenium, arsenic, cadmium, and lead); poultry by-product meals (zinc); shellfish (zinc); seleniferous accumulating plants of the genera Astragalus and Machaeranthera, or cereals grown in seleniferous soils (selenium). Excessive minerals may cause chronic poisoning, inhibition of enzyme activity in shrimp, and environmental pollution. Moreover, contamination with minerals of the shrimp body may harm human health. Thus we must ascertain requirement for shrimp carefully, and choose the high bioavailability form.

With dietary Cu supplementation increased, weight gain ratio, feed efficiency and specific growth rate first increased and then decreased. Moreover, higher copper supplement level (?100mg/kg) has toxicity to P. vannamei (Liu et al., 2008). It was demonstrated in P. monodon that the higher shrimp mortality associated with the lower non-specific immune responses caused by excess Cu (?80mg/kg) (Lee and Shiau, 2002a).

Minerals and immune responses

Some minerals have been proved to have influence on immune responses of fish, for example phosphorus, iron, copper, zinc and selenium. However, reference on effects of dietary minerals on immune responses and disease resistance in shrimp is limited, and published data mainly focus on P. monodon fed with dietary copper or zinc. Hereby, we present some information about effects of dietary copper and zinc on non-specific immune responses of shrimp.

Copper

There is a close relationship between the dietary Cu level, activity of enzymes and cellular and immune functions. Copper deficiency in human subject is reflected in the low Cu content in leucocytes. It functions in hematopoiesis and in numerous Cu-dependent enzymes including lysyl oxidase, cytochrome c oxidase (CCO), ferroxidase, tyrosinase and superoxide dismutase (SOD). Information about relationship between dietary Cu and immune responses in shrimp is limited. Lee and Shiau (2002a) reported that non-specific immune responses in shrimp P. monodon fed diets containing various levels of Cu support the weight gain data, in that groups of shrimp fed diets containing 10–30 mg Cu/kg diet had the highest total hemocyte count and intracellular superoxide anion production ratio. Collectively, the adequate dietary Cu requirement for maintenance of non-specific immune responses of P. monodon reared in brackish water containing 1.53 ?g Cu/l water is about 10–30 mg Cu/kg diet.

Zinc

The primary functions of zinc are based on its roles as a cofactor in several enzyme systems and as a component of a large number of metalloenzymes, including carbonic anhydrase, alkaline phosphatase, carboxypeptidase, alcohol dehydrogenase, glutamic dehydrogenase, lactate dehydrogenase, ribonuclease, and DNA polymerase. As zinc is needed to maintain normal activity of lymphocytes, making it essential for the integrity of the immune system, its supplementation to diets may improve immune function. Information on the relationship between dietary zinc and immune responses in shrimp is limited. Shiau and Jiang (2006) fed P. monodon with graded dietary zinc, and found that both intracellular superoxide anion (O2-) production ratios and total hemocyte count (THC) were highest in shrimp fed diets with 35 and 48 mg Zn/kg. Furthermore, they concluded that an adequate dietary Zn concentration for nonspecific immune responses in P. monodon is about 35–48 mg Zn/kg diet.

NON-NUTRITIONAL IMMUNOSTIMULANTS

Culture conditions promoting chronic, subacute, or acute disease outbreaks include crowding, handling, accumulation of wastes, ambient flora and fauna, oxygen levels, exposure to sunlight, and water temperature. Crustaceans lack a truly adaptive immune response system and appear to rely on a variety of innate immune response systems to rapidly and efficiently recognize and destroy “non-self” materials (Lee and Söderhäll, 2002). Unlike vaccines, immunostimulants simultaneously elevate the overall resistance of animals to many infectious agents by stimulating the nonspecific immune response. Immunostimulants are promoted in aquaculture as a means to overcome the immunosuppressive effects that occur in normal aquaculture operations, due to stressors or unavoidable consequences of high-density culture. They might be used as a prophylactic treatment in anticipation of expected seasonal outbreaks of known endemic diseases or a suppressive treatment for latent or sublethal pathogens.

Immunostimulants are widely divided into two classes: nutritional immunostimulants and non-nutritional immunostimulants. The former include vitamins (e.g., vitamin C, B6, E and A), minerals (e.g., selenium, zinc and copper), polyunsaturated fatty acids (e.g., n-3 fatty acids), etc. Sources of the latter may be chemically or biologically produced (Sakai, 1999). The biological sources include polysaccharides, oligosaccharides, microecological preparation and herbs (e.g., Chinese herbal medicine), etc. The chemical sources include levamisole, FK-565 (heptanoyl-y-D-glutamyl-(L)-meso- diaminopimelyl-(D)-alanine), etc.

The use of immunostimulants as diet additives has met with uncertainty and skepticism because the actions of these compounds are poorly understood and the results of feeding trials have not shown consistently positive effects. Some products are of unclear nature and origin. The exact mechanism of immunostimulant absorption is not known. However, they have been widely accepted by shrimp farmers.

Polysaccharides

Most of the immunostimulants in aquaculture diets are polysaccharides derived from bacteria, fungi or yeasts, and plants. The substances may be the cells themselves or preparations from the cell walls. An emphasis in fisheries research has been placed on ß-glucans, preferred in aquaculture because they occur naturally and are less likely to cause concerns about residue in food shrimp or water quality.

ß-Glucans

ß-Glucans are glucose polymers found in the cell walls of yeast, fungi and cereal plants. It consists of a backbone of ß-(1?3)-linked ß-D-glucopyranosyl units with ß-(1?6)-linked side chains of varying length and distribution. The effect of ß-glucan has been attributed to its binding to several receptors on leucocytes resulting in the stimulation of immune responses, such as bacteria killing activity, modulation of cytokine production and survival promotion at the cell, organ and whole animal levels.

Currently many commercial immunostimulants are available in the shrimp aquaculture industry and are extensively used by shrimp farmers. However, scientific data in support of their function and dose/frequency of application are lacking. Itami et al. (1998) reported that dietary administration of ß-glucan derived from Bifidobacterium thermophilum increased the resistance of P. japonicus against vibriosis. Using a different glucan, ß1-3/1-6 glucan extracted from the yeast cell wall, Sung et al. (1998) demonstrated enhanced resistance of P. monodon to vibriosis and white spot syndrome virus (WSSV) infection. Chang et al. (2000) showed that ß1-3 glucan enhanced hemocyte phagocytic activity, cell adhesion, and superoxide anion production when added to P. monodon diets. Tan et al. (2004) suggested that the oral administration of ß1-3/1-6 glucan to P. vannamei improved growth, enhanced the immunity and increases disease resistance. Moreover, they pointed out that 0.1 % of ß1-3/1-6 glucan was recommended for enhancing the immunity as well as increase disease resistance and 0.2 % of ß1-3/1-6 glucan for improving growth of P. vannamei (Tan et al., 2004).

In addition to the dose of glucan, the feeding frequency is also important for application of glucan in shrimp feeds. In the research of Sajeevan et al. (2009), the immunostimulatory effect of an alkali insoluble glucan extracted from marine yeast isolate Candida sake S165 was tested in P. indicus. Post larvae (PL) of P. indicus, fed glucan incorporated diet at varying concentrations (0.05, 0.1, 0.2, 0.3, 0.4 g glucan/100 g feed) for 21 days were challenged orally with white spot syndrome virus (WSSV). Maximum survival was observed in PL fed the 0.2% glucan incorporated diet. Subsequently the feed incorporated with 0.2% glucan was fed to P. indicus post larvae at different feeding intervals, i.e. daily, once every two days, once every five days, once every seven days and once every ten days. After 40 days, the shrimps were challenged orally with WSSV and post challenge survival was recorded. Shrimp fed containing 0.2% glucan when administered once every seven days gave maximum survival. This was supported by hematological data obtained from adult P. indicus, i.e. total hemocyte count, phenoloxidase activity and nitroblue tetrazolium reduction (NBT). These data confirmed the importance of dose and frequency of administration of immunostimulants in shrimp health management.

Lipopolysaccharide

Lipopolysaccharide (LPS) is derived from the outer cell wall membrane of Gram-negative bacteria. It is usually large molecular weight substances with unique chemical structure, consisting of three regions, i.e., an outer polysaccharide region (commonly known as ‘O’ antigen, a linear or branched component of oligosaccharide residues), a unique polysaccharide core region (consisting of short chain sugars), and an inner fatty acid rich region (i.e., Lipid A containing diglucosamine unit with long-chain fatty acids). Higher animals are extremely sensitive to endotoxin/LPS even at low doses but lower vertebrates such as frog and fish are stated to be resistant to endotoxic shock. However, endotoxin/LPS has been found many times to be responsible for the pathogenicity of several bacterial diseases, especially of Gram-negative origin, in shrimp.

Sritunyalucksana et al. (1999) pointed out that LPS was in vitro involved in mechanisms for both clotting and for antibacterial activity in the black tiger prawn P. monodon. Takahash et al. (2000) demonstrated that oral administration of LPS enhanced the disease resistance against penaeid acute viraemia and inducted virus-inactivating activity in hemolymph of shrimp P. japonicus. In the studies of the P. chinensis it was found that after stimulated with LPS, the number of the overall hemocytes increased 83.4 %, in which the semigranular cells increased 100.4 %, and the granular cells increased 47 % (Wang et al., 2005). It was shown that the southern white shrimp Litopenaeus shmitti is able to recognize LPS of E. coli and to respond to this microbial elicitor by the variation in the levels of the phenoloxidase activity in plasma, the total hemocyte counts and the levels of nitric oxide (Rodríguez-Ramos et al., 2008). Okumura (2007) suggested that the antimicrobial peptide system (penaeidins and crustin) in Pacific white shrimp P. vannamei responded to LPS injection at a gene expression level while the prophenoloxidase system did not respond at a gene expression level.

Other polysaccharides

There are also some other polysaccharides proved to be immunostimulants for aquaculture animals. Researches from Itami et al. (1998) indicated that the oral administration of peptidoglycan derived from Bifidobacterium thermophilum enhanced the phagocytic activity of the granulocytes and increased the disease resistance of P. japonicus. Wang and Chen (2005) concluded that P. vannamei that received chitin at 6 ?g/g or chitosan at 4 ?g/g or less increased its immune ability and resistance to V. alginolyticus infection. Huang et al. (2006) suggested that oral administration of Sargassum fusiforme polysaccharide extracts at an optimal level of 0.5% and 1.0% for 14 days effectively improved vibriosis resistance and enhanced immune activity of the shrimp P. chinensis in general.

Probiotics

Probiotics are defined as live microbial or cultured product feed supplements, which beneficially affect the host by producing inhibitory compounds, competing for chemicals and adhesion sites, modulating and stimulating the immune functions, and improving the microbial balance. In aquaculture, probiotic can be administered either as a food supplement or as an additive to the water as a means of controlling disease, enhancing the immune response, supplementing or even in some cases replacing the use of antimicrobial compounds, providing nutrients and enzymatic contributions, and improving water quality. A wide range of microalgae (Tetraselmis), yeasts (Debaryomyces, Phaffia, and Saccharomyces), and Gram-positive (Bacillus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Streptococcus, and Weissella) and -negative bacteria (Aeromonas, Alteromonas, Photorhodobacterium, Pseudomonas, and Vibrio) have been applied as probiotics to improve aquatic animal growth, survival, health, and disease prevention (Son et al., 2009).

Most studies on probiotic in shrimp culture conducted in P. vannamei. Four bacterial strains were isolated from the gastrointestinal tract of adult shrimp P. vannamei, Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Pseudomonas aestumarina SLV22. Subsequently, they are demonstrated to be probiotics for P. vannamei to resist disease caused by V. parahaemolyticus (Balcázar et al., 2007). There are also some other probiotics for P. vannamei either as a supplement in diet or as an additive in aquaculture water. They are Bacillus OJ isolated from the digestive tracts of P. vannamei (Li et al., 2009), B. coagulans SC8168 obtained from the pond sediment (Zhou et al., 2009), B. subtilis E20 isolated from fermented soybean (Tseng et al., 2009), etc.

Herbs

Chinese herbs have been used as traditional medicines and immune boosters for human beings for thousands of years in China. These herbs contain many types of active components, like polysaccharides, alkaloids or flavonoids. The immunostimulating activity of herbal components has been most widely studied in mice, chickens or human cell lines. Recently, growing interest has been paid to the immune stimulating function of some herbs in aquaculture.

Chinese herbs proved to be able to improve the immunity of fish and shellfish are as follows: (1) to fish, the herbs are Rheum officinale, Andrographis paniculata, Isatis indigotica, Lonicera japonica (Chen et al., 2003), Astragalus radix and Ganoderma lucidum (Yin et al., 2009), Radix astragalin seu Hedysari and R. Angelicae Sinensis (Jian and Wu, 2004), etc.; (2) to abalone, a traditional Chinese medicine preparation was formulated from orange peel (Pericarpium Citri Reticulatae), hawthorn (Crataegus pinnatifida), astragalus (Astragalus membranaceus (Fisch.) Bunge), pilose asiabell root (Radix codonopsis), indigowoad root (Radix isatidis), taraxacum (Herba taraxaci) and malt (Fructus Hordei Germinatus) at a weight ratio of 1:1:1.5:1.5:1.5:1.5:2 (Xue et al., 2008).

In shrimp, Lin et al. (2006) fed P. vannamei with diets supplemented graded levels of a mixture of equal proportions of six herbs (Isatis tinctoria L., Isatis indigodica Fort, Forsythia suspersa Vahl, Corydalis bungeana Turez, Pogostemon cablin (Blanco) Benth and Astragalus menbranaceus (Fisch.) Bge). They found that apparent digestibility coefficients of crude protein decreased as herbs increased. However, apparent digestibility coefficients of lipid and phosphorus increased as herbs increased. Yeh et al. (2008) suggested that the extracts of stout camphor tree could be a candidate to replace the chemo-therapeutants through the inhibitory effects against the growth of pathogens, and enhanced effects on shrimp P. vannamei immunity and disease resistance.

There are also some published data indicate that some Indian herbs have been proved to be able to improve the immunity of shrimp. Terrestrial plant Ricinus communis could significantly improve the ability of P. indicus to resist the challenge from pathogen Vibrio parahaemolyticus (Immanuel et al., 2004). Based on the data of influence of selected Indian immunostimulant herbs (Cyanodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba) against WSSV infection in P. monodon, Citarasu et al. (2006) revealed that the application of herbal immunostimulants will be effective against shrimp viral pathogenesis.

Acknowledgments

The authors would like to thank the collaborators in the Laboratory of Aquaculture Nutrition and Feeds, Ocean University of China. They are Xiaoru Chen, Yi Zhou, Chenglong Wu, Jie Chang and Nan Bai. We also thank Dr. Allen Davis from Auburn University of the United States for his constructive suggestions.

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